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Science AdvancesCell BiologyPeer-reviewed journal article

Type-II kinase inhibitors that target Parkinson's Disease-associated LRRK2

Nicolai D. RaigiD, Katherine J. SurridgeiD, Marta M. Sanz MurilloiD, Verena DedereriD, Andreas KrämeriD, Martin P. SchwalmiD, Nicholas M. LattaliD, Lewis ElsoniD, Deep ChatterjeeiD, Sebastian MatheaiD, Thomas HankeiD, Andrés E. LeschzineriD, Samara L. Reck‐PetersoniD, Stefan KnappiD

PDFDOIPreprintData

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (13 Feb 2025) · CC BY 4.0

Hybrid type-II inhibitors RN277 and RN341 target inactive LRRK2 with far fewer kinase off-targets than Rebastinib, preserve S935 phosphorylation, and rescue kinesin motility in vitro.

Published selective LRRK2 inhibitors are all type-I, which stabilize the closed conformation, and the available type-II inhibitors are broad-spectrum. This work builds LRRK2-selective type-II compounds by fusing type-I hinge binders with type-II back-pocket groups, to probe the inactive conformation.

Key results

  1. ShownIterative hybridization of type-I hinge binders (MLi-2 and PF-360 scaffolds) with type-II back-pocket groups produced RN277 and RN341. Most early MLi-2 hybrids bound LRRK2 poorly; the potent early hybrids (52, 2, RN129) were promiscuous.

    MLi-2 hybrid 52: ΔTm 10 K, sub-micromolar IC50, but ΔTm shifts for 27 % of ~100 kinases. RN129: IC50 59.7 nM, 49 % of kinases stabilized. RN277: S(5K) = 10 %, ΔTm 18 K, IC50 135 nM. RN341: ΔTm 20 K, IC50 296 nM, S(5K) = 10 %. · MLi-2 hybridization series; PF-360 hybridization series; PF-360-Rebastinib hybrid series

  2. ShownA cryo-EM structure of LRRK2 RCKW with RN277 shows a type-II binding mode with an open, inactive kinase. It differs in several details from the GZD-824 complex.

    Overall resolution 3.3 Å (about 3 Å near the active site). DYG out, αC out, broken R-spine, disordered activation loop. K1906 to E1920 distance 5.1 Å vs 3.4 Å with GZD-824. G-rich loop not resolved, and H1998 has a single rotamer. One structure, for RN277 only. · RN277 is a type-II inhibitor of LRRK2

  3. ShownRN341 is much more selective than its parent Rebastinib. Only a few off-targets reach micromolar cellular potency.

    DSF: 37 kinases shifted by >5 K with Rebastinib vs 10 with RN341. 350-kinase activity screen at 10 µM: 10 kinases below 30 % residual activity; at 1 µM only TTK below 50 %. nanoBRET: 4 of 12 off-targets with EC50 < 5 µM (STK10 1.5, MAPK14 1.7, JNK2 2.7, TTK 3.2 µM). · PF-360-Rebastinib hybrid series; Kinome selectivity profiling of RN341; Figure legends

  4. ShownBoth compounds inhibit LRRK2 phosphorylation of Rab8a in vitro and in 293T cells, without the S935 dephosphorylation seen with the type-I inhibitor MLi-2.

    Mass spectrometry IC50 on Rab8a: 70 nM (RN277) and 110 nM (RN341). At 5 µM in cells, pRab8a fell to near background, as with 500 nM MLi-2. Only MLi-2 markedly reduced pS935. Four independent western blots, one-way ANOVA. · In vitro and in cellulo inhibition of LRRK2…; Figure legends

  5. SuggestedRN277 and RN341 rescue kinesin motility blocked by LRRK2 RCKW in single-molecule assays, like the type-II inhibitor Ponatinib, whereas MLi-2 does not.

    TIRF assay with K560-GFP kinesin; inhibitors at 5 µM; LRRK2 RCKW reduced the percent of motile events in the DMSO (p = 0.0007) and MLi-2 (p = 0.0003) conditions. The Fig. 5E legend gives ***p 0.0003 for the type-II panel without specifying the comparison. Three technical replicates per condition. · Type-II inhibitors rescue LRRK2 inhibited…; Single-molecule motility assays; TIRF motility data analysis; Figure legends

Methods, data and reproducibility

Hybrid compounds were designed from type-I hinge binders and type-II back-pocket groups, then tested by thermal shift (DSF), enzymatic IC50, kinome selectivity and nanoBRET assays. The leads were characterized by cryo-EM, Rab8a phosphorylation in vitro and in cells, and kinesin motility assays.

  • Cryo-EM of LRRK2 RCKW bound to RN277 and DARPin E11 (13,972 movies, 412,439 particles) · n = 1 structure, 3.3 Å · EMD-47004, EMD-47006, EMD-47025; PDB 9DMI
  • Crystal structure of CLK3 kinase domain bound to RN129 · n = 1 structure · PDB 9EZ3
  • DSF selectivity panels (~100 kinases; 103 for RN341, 96 for Rebastinib) · n = approximately 100 kinases per compound
  • 33PanQinase activity screen of RN341 at 1 and 10 µM · n = 350 kinases
  • nanoBRET off-target validation in HEK293T cells · human (HEK293T cell line) · n = 12 off-target kinases; 2 biological replicates
  • Rab8a phosphorylation western blots in 293T cells co-transfected with LRRK2 and GFP-Rab8a · human (293T cell line) · n = 4 independent blots
  • Single-molecule kinesin motility assays on microtubules · n = 3 technical replicates per condition
Methods & tools
Differential scanning fluorometry (DSF), PhosphoSense assay (AssayQuant), 33PanQinase (Reaction Biology), NanoBRET, CryoSPARC 4.3, Phenix 1.21, Coot, ColabFold, UCSF ChimeraX, ImageJ, GraphPad Prism, XDS
Reagents & environment
CryoSPARC 4.3, Phenix 1.21, UCSF ChimeraX 1.7, Coot 0.8.9, ImageJ 2.14.0, Topaz 0.2.5, GraphPad Prism 8/9/10.
Reproducibility gaps
No analysis code is listed. Synthesis procedures, compound tables S1 and S2, and kinome data S1 are in the Supporting Information, not in the text provided. The CLK3 diffraction dataset is on Zenodo (https://doi.org/10.5281/zenodo.13934795).
Limitations (4)
  • RN341 was not detected as a LRRK2 inhibitor in the 350-kinase activity panel. The authors attribute this to LRRK2 being large and unstable in vitro, so the kinase was likely inactive there, and rely on other assays. · stated by the authors
  • Only RN277 was solved by cryo-EM. The authors anticipate that RN341 binds in a largely comparable manner, based on structural and chemical similarity. · stated by the authors
  • Selectivity rests mainly on thermal-shift panels of about 100 kinases plus one activity screen. Cellular validation covers only 12 selected off-targets, and no in vivo or pharmacokinetic data are shown. · noted in this summary
  • The motility rescue comes from in vitro assays with three technical replicates per condition. Cellular effects on LRRK2 filament formation or lung pathology are not tested. · noted in this summary

Read this if you need a selective chemical probe for the inactive, DYG-out conformation of LRRK2, or want a worked example of hybridizing type-I and type-II kinase inhibitors.

Citations
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Abstract

Aberrant increases in kinase activity of leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinsons disease (PD). Numerous LRRK2-selective type-I kinase inhibitors have been developed and some have entered clinical trials. In this study, we present the first LRRK2-selective type-II kinase inhibitors. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type-I inhibitors. We designed these inhibitors using a combinatorial chemistry approach fusing selective LRRK2 type-I and promiscuous type-II inhibitors by iterative cycles of synthesis supported by structural biology and activity testing. Our current lead structures are selective and potent LRRK2 inhibitors. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2s function and regulation, and expand the potential therapeutic options for PD.

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